Dopa decarboxylase inhibitor compositions

ABSTRACT

Disclosed herein are formulations containing carbidopa and optionally levodopa, arginine, and other components that have reduced levels of impurities and toxins, particularly degradation productions. Also disclosed herein are methods of treatment diseases or conditions relating to a loss of dopamine or dopaminergic neurons using such formulations, methods of making such formulations, and kits that include such formulations.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/438,472, filed Feb. 21, 2017, which is a continuation-in-part of U.S.patent application Ser. No. 14/645,848 filed Mar. 12, 2015 (now U.S.Pat. No. 10,022,320), which in turn claims the benefit of and priorityto U.S. Provisional Application No. 61/952,477, filed Mar. 13, 2014, andU.S. Provisional Application No. 61/990,967, filed May 9, 2014, each ofwhich is hereby incorporated by reference.

BACKGROUND

Parkinson's disease is a degenerative condition characterized by reducedconcentration of the neurotransmitter dopamine in the brain. Levodopa(L-dopa or L-3,4-dihydroxyphenylalanine) is an immediate metabolicprecursor of dopamine that, unlike dopamine, is able to cross theblood-brain barrier and is most commonly used for restoring the dopamineconcentration in the brain. For the past 40 years, levodopa has remainedthe most effective therapy for the treatment of Parkinson's disease.

However, levodopa has a short half-life in plasma that, even under bestcommon current standard of care, results in pulsatile dopaminergicstimulation. Long-term therapy is therefore complicated by motorfluctuations and dyskinesia that can represent a source of significantdisability for some patients. A therapeutic strategy that couldultimately deliver levodopa/dopamine to the brain in a more continuousand physiologic manner would provide the benefits of standard levodopawith reduced motor complications and is much needed by patientssuffering from Parkinson's disease and other neurological or movementdisorders (Olanow C W; Mov. Dis. 2008, 23 (Suppl. 3):5613-5622).Sustained-release oral levodopa formulations have been developed, but,at best, such preparations have been found to be no more efficaciousthan standard tablets. Continuous administration of levodopa byintraduodenal administration or infusion has also been attempted byusing ambulatory pumps or patches. Such treatments, especiallyintraduodenal, are extremely invasive and inconvenient.

The metabolic transformation of levodopa to dopamine is catalyzed by thearomatic L-amino acid decarboxylase enzyme, a ubiquitous enzyme withparticularly high concentrations in the intestinal mucosa, liver, brain,and brain capillaries. Due to the possibility of extracerebralmetabolism of levodopa, it is necessary to administer large doses oflevodopa leading to high extracerebral concentrations of dopamine thatcause nausea in some patients. Therefore, levodopa is usuallyadministered concurrently with oral administration of a dopadecarboxylase inhibitor, such as carbidopa or benserazide, which reducesby 60-80% the levodopa dose required for a clinical response, and thusprevents certain of its side effects by inhibiting the conversion oflevodopa to dopamine outside the brain.

Various oral formulations together with inhibitors of enzymes associatedwith the metabolic degradation of levodopa are well known, for example,decarboxylase inhibitors such as carbidopa and benserazide, monoamoneoxidase (MAO)-A or MAO-B inhibitors such as moclobemide, rasagiline,selegiline, and safinamide, and catechol-O-methyl transferase (COMT)inhibitors such as tolcapone and entacapone. Currently available oraldrugs include SINEMET® and SINEMET®CR sustained-release tablets thatinclude carbidopa or levodopa; MADOPAR® tablets containing levodopa andbenserazide; and STALEVO® tablets containing carbidopa, entacapone, andlevodopa.

Carbidopa is a non-competitive inhibitor of DOPA decarboxylase. Whenmixed with levodopa, carbidopa inhibits the peripheral conversion oflevodopa to dopamine. This results in increased levodopa available fortransport to the CNS. Carbidopa also inhibits the metabolism of levodopain the GI tract, thus, increasing levodopa bioavailability. It is usedin Parkinson's disease to reduce the peripheral effect of dopamine. Theloss of the hydrazine functional group represents the major metabolicpathway for carbidopa.

Hydrazine (N₂H₄), or its salts, are used in the production ofpharmaceutical products. It has been the cause of severe adverse effectson the central nervous system, liver, and kidneys. In addition to theseeffects, experimental animals have also shown the following symptoms:loss of body weight, anaemia, hypoglycaemia, fatty degeneration of theliver, and convulsions. Hydrazine has also been shown to cause DNAdamage, gene mutations, and chromosome aberrations (Environmental healthcriteria No. 68 hydrazine (1987)) and has induced tumor growth in mice,hamsters, and rats after oral, intraperitoneal, and inhalationadministration (MacEwan J D, Vernot E H, Haun C C, et al. (1981)).Hydrazine and its salts are used in the pharmaceutical industry as anintermediate to produce drugs with different therapeutic effectsincluding decarboxylase inhibitiors, antihypertensives, andantibacterials. Since hydrazine is toxic and thought to be a possiblehuman carcinogen, its presence is limited in some of these drugsubstances in the monographs of the European Pharmacopoeia (Ph. Eur.).

Accordingly, there is an ongoing and urgent need for liquid formulationsand compositions that can achieve continuous dopaminergic stimulation totreat movement disorders such as Parkinson's disease more effectivelycontaining a safe and tolerable amount of hydrazine.

SUMMARY

This disclosure is directed in part to carbidopa or carbidopa esterformulations (CD), which can include levodopa (e.g., includes levodopa(or a levodopa ester) and carbidopa). In certain embodiments, disclosedcarbidopa or carbidopa/levodopa formulations also include two or moreantioxidants, e.g., (a) ascorbic acid or a salt thereof (e.g., sodiumascorbate) and (b) another antioxidant, such as cysteine or a cysteinederivative (for example, L-cysteine or N-acetylcysteine (NAC),glutathione, or diacetylcystine), or a sulfite (e.g., sodium sulfite).In particular, we have discovered that CD formulations that include twoantioxidants are more stable than those containing just a singleantioxidant. Certain disclosed compositions or formulations haveimproved resistance to degradation (e.g., have minimal amounts of adegradation species, e.g., hydrazine), and/or are significantly stable.

Accordingly, in a first aspect, the invention contemplates apharmaceutically acceptable formulation. In one embodiment, theformulation includes levodopa, about 0.1% to about 6% by weightcarbidopa, about 1% to about 25% by weight of a component selected fromthe group consisting of arginine, meglumine, and a combination thereof,and at least one o-quinone scavenger. In another embodiment, theformulation includes about 8% to about 16% (e.g., about 11% to 15% orabout 12% to about 14%) by weight levodopa, about 1% to about 4% byweight carbidopa, about 0.1% to about 40% by weight of a componentselected from arginine, meglumine, and a combination thereof, and atleast one o-quinone scavenger. In either of these embodiments, thepharmaceutically acceptable formulation can have less than about 20, 10,5, 2 1.0, 0.75, 0.5, 0.25, 0.1, 0.05, or 0.025 μg/ml of hydrazine, e.g.,as determined by a gas chromatography-mass spectrometry (GCMS) method.In particular embodiments, the formulation has less than about 10 or 5or 2 or 1 or 0.1 or 0.05 μg/ml of hydrazine or about 0.1 to about 0.5μg/ml, or about 20 to about 0.1 μg/ml, about 10 to about 0.5 μg/ml, orabout 1 to about 0.5 μg/ml of hydrazine, e.g., as determined by a GCMSmethod.

The formulation may include an o-quinone scavenger selected from thegroup consisting of ascorbic acid or a salt thereof, L-cysteine, NAC,glutathione, diacetylcystine and/or a salt thereof, and a combinationthereof. The formulation may further include about 0.1% to about 10% byweight ascorbic acid or a salt thereof and a component selected from thegroup consisting of about 0.01% to about 1% by weight of NAC, about0.01% to about 1% by weight L-cysteine, about 0.001% to about 1% byweight glutathione, and about 0.001% to about 1% by weightdiacetylcystine or a salt thereof.

In another aspect, the invention contemplates a pharmaceuticallyacceptable formulation including (a) carbidopa (e.g., about 0.1% toabout 10% carbidopa); (b) ascorbic acid or a salt thereof; and (c) oneof L-cysteine, NAC, glutathione, and diacetylcystine, or a salt thereof.The formulation may include less than 20, 10, 5, 2, 1.0, 0.75, 0.5,0.25, 0.1, 0.05, or 0.025 μg/ml of hydrazine, e.g., as determined by aGCMS method. In particular embodiments, the formulation has less thanabout 5 μg/ml of hydrazine, less than about 0.1 μg/ml of hydrazine, lessthan about 0.05 μg/ml of hydrazine, or about 0.1 to about 0.5 μg/ml ofhydrazine, e.g., as determined by a GCMS method. The formulation mayinclude about 0.1% to 10% (e.g., 0.3% to about 2%, about 0.5%, about1.0% to about 1.3%, about 1.2%, or about 1.3%) by weight ascorbic acid.The formulation may include about 0.01% to about 1% (e.g., about 0.1% toabout 0.6%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, or about0.8%) by weight L-cysteine, or a salt thereof. The formulation mayinclude about 0.1% to about 10% (e.g., about 0.1% to about 6%, about0.1% to about 4%, about 0.6% to about 1.4%, about 1.2% to about 4%,about 0.75%, about 1.4%, about 3%, or about 3.3%) by weight carbidopa.The formulation may include about 0.1% to about 10% (e.g., about 0.4% toabout 0.6%, about 0.4% to about 1%, about 0.5%, or about 1.2%) by weightascorbic acid, or a salt thereof. The formulation may include about0.01% to about 1% (e.g., about 0.1% to about 1%, about 0.3%, about 0.4%,about 0.5%, about 0.6%, about 0.7%, or about 0.8%) by weight L-cysteineor NAC. The composition may include less than 4% (e.g., less than 2%,1%, 0.5%, 0.1%, 0.05%, or 0.01%) by weight levodopa or may not includelevodopa. In certain embodiments, the composition includes levodopa(e.g., about 2% to about 16%, about 2% to about 8%, about 8% to about16%, about 6%, about 12% to about 15%; about 2% to about 16%, about 12%,or about 13% by weight levodopa). The composition may further comprisearginine, meglumine, or a combination thereof, for example, about 0.1%to about 40%, about 1% to about 25%, about 10% to about 25%, about 12%to about 40%, about 32% to about 42%, or about 15% to about 16% byweight arginine, meglumine, or a combination thereof. The formulationmay include the components in the following Table:

Components Exemplary Amount Exemplary amount Levodopa  0-16%    5-7%Carbidopa  0.1-6% 0.6-1.5% Arginine 0.1-40%  14-16% Ascorbic acid or Na0.1-10% 0.3-0.7% ascorbate L-cysteine or NAC or 0.01-1% 0.3-0.5%glutathione

In particular embodiments, the formulation includes about 2% to about 8%by weight levodopa, about 0.1% to about 3% by weight carbidopa, about10% to about 25% by weight arginine, about 0.1% to about 10% (e.g.,about 0.3% to about 2%) by weight ascorbic acid or salts thereof, andabout 0.001% to about 5% by weight L-cysteine or a salt thereof. Inother embodiments, the formulation includes about 8% to about 16% byweight levodopa, about 1% to about 4% by weight carbidopa, about 12% toabout 40% by weight of a component selected from the group consisting ofarginine, meglumine, and a combination thereof, about 0.1% to about 10%by weight ascorbic acid or a salt thereof, and about 0.001% to about 1%by weight L-cysteine or a salt thereof. In either of these embodiments,the formulation has less than about 5, 2, 1, 0.5 or 0.1 μg/ml hydrazine(e.g., less than 0.05 or 0.01 μg/ml hydrazine), as determined by GCMS.The formulation may include the components in the following Tables:

Components Exemplary Amount Exemplary amount Levodopa  4-8%    5-7%Carbidopa 0.5-2% 0.6-1.5% Arginine 13-18%   14-16% Ascorbic acid 0.1-2%0.3-0.7% L-cysteine or cysteine HCl 0.1-2% 0.3-0.5%

Components Exemplary Amount Exemplary amount Levodopa 10-15%   12-15%Carbidopa 1.2-4%    2-4% Arginine/meglumine or a 25-40%   30-38%combination thereof Ascorbic acid or sodium 0.1-2% 0.3-0.7% ascorbateL-cysteine or cysteine-HCl 0.1-1% 0.2-0.5%

In other particular embodiments, the formulation includes about 2% toabout 8% by weight levodopa, about 0.1% to about 3% by weight carbidopa,about 10% to about 25% by weight arginine, about 0.1% to about 10% byweight ascorbic acid or a salt thereof, and about 0.001% to about 5% byweight NAC. In other embodiments, the formulation includes about 8% toabout 16% by weight levodopa, about 1% to about 4% by weight carbidopa,about 12% to about 40% by weight of a component selected from the groupconsisting of arginine, meglumine, and a combination thereof, about 0.1%to about 10% by weight ascorbic acid or a salt thereof, and about 0.001%to about 1% by weight NAC. In either of these embodiments, theformulation has less than about 5 or 2 or 0.5 or 0.1 μg/ml hydrazine(e.g., less than 0.05 or 0.01 μg/ml hydrazine), as determined by GCMS.The formulation may include the components in the following Tables:

Components Exemplary Amount Exemplary amount Levodopa  4-8%    5-7%Carbidopa 0.5-2% 0.6-1.5% Arginine 13-18%   14-16% Ascorbic acid 0.1-2%0.3-0.7% NAC 0.1-2% 0.3-0.5%

Components Exemplary Amount Exemplary amount Levodopa 10-15%   12-15%Carbidopa 1.2-4%    2-4% Arginine/meglumine or a 25-40%   30-38%combination thereof Ascorbic acid or sodium 0.1-2% 0.3-0.7% ascorbateNAC 0.1-1% 0.2-0.5%

In particular embodiments, the formulation includes about 2% to about 8%by weight levodopa, about 0.1% to about 3% by weight carbidopa, about10% to about 25% by weight arginine, about 0.1% to about 10% by weightascorbic acid or a salt thereof, and about 0.001% to about 5% by weightglutathione. In other embodiments, the formulation includes about 8% toabout 16% by weight levodopa, about 1% to about 4% by weight carbidopa,about 12% to about 40% by weight of a component selected from the groupconsisting of arginine, meglumine, and a combination thereof, about 0.1%to about 10% by weight ascorbic acid or a salt thereof, and about 0.001%to about 1% by weight glutathione. In either of these embodiments, theformulation has less than about 5, 2, 0.5 or 0.1 μg/ml hydrazine (e.g.,less than 0.05 or 0.01 μg/ml hydrazine), as determined by GCMS. Theformulation may include the components in the following Tables:

Components Exemplary Amount Exemplary amount Levodopa  4-8%    5-7%Carbidopa 0.5-2% 0.6-1.5% Arginine 13-18%   14-16% Ascorbic acid 0.1-2%0.3-0.7% Glutathione 0.1-2% 0.3-0.5%

Components Exemplary Amount Exemplary amount Levodopa 10-15%   12-15%Carbidopa 1.2-4%    2-4% Arginine/meglumine or a 25-40%   30-38%combination thereof Ascorbic acid or sodium 0.1-2% 0.3-0.7% ascorbateGlutathione 0.1-1% 0.2-0.5%

In particular embodiments, the formulation includes about 2% to about 8%by weight levodopa, about 0.1% to about 3% by weight carbidopa, about10% to about 25% by weight arginine, about 0.1% to about 10% by weightascorbic acid or a salt thereof, and about 0.001% to about 5% by weightdiacetylcystine or a salt thereof. In other embodiments, the formulationincludes about 8% to about 16% by weight levodopa, about 1% to about 4%by weight carbidopa, about 12% to about 40% by weight of a componentselected from the group consisting of arginine, meglumine, and acombination thereof, about 0.1% to about 10% by weight ascorbic acid,and/or a salt thereof, and about 0.001% to about 1% by weightdiacetylcystine or a salt thereof. In either of these embodiments, theformulation has less than about 5, 0.5 or 0.1 μg/ml hydrazine (e.g.,less than 0.05 or 0.01 μg/ml hydrazine), as determined by GCMS. Theformulation may include the components in the following Tables:

Components Exemplary Amount Exemplary amount Levodopa  4-8%    5-7%Carbidopa 0.5-2% 0.6-1.5% Arginine 13-18%   14-16% Ascorbic acid 0.1-2%0.3-0.7% Diacetylcystine 0.1-2% 0.3-0.5%

Components Exemplary Amount Exemplary amount Levodopa 10-15%   12-15%Carbidopa 1.2-4%    2-4% Arginine/meglumine or a 25-40%   30-38%combination thereof Ascorbic acid or sodium 0.1-2% 0.3-0.7% ascorbateDiacetylcystine 0.1-1% 0.2-0.5%

The formulation of any of the above embodiments may include asurfactant. The surfactant may be any of one of polysorbate 20, 40, 60,and 80, or a combination thereof. In particular embodiments, theformulation includes about 0.01% to about 5% surfactant (e.g.,polysorbate 80) or about 0.1% to 0.5% surfactant (e.g., polysorbate 80).In more particular embodiments, the formulation includes about 0.3%surfactant (e.g., polysorbate 80).

The formulation of any of the above embodiments may include about 11% toabout 15% by weight levodopa. For example, the formulation may includeabout 12% to about 14% by weight levodopa (e.g., about 12% or about13.2% levodopa).

The formulation of any of the above embodiments may include about 0.6%to about 4%, or 0.8% to about 3%, or about 1.2% to about 4%, by weightcarbidopa. For example, the formulation may include about 2.5% to about3.5% (e.g., about 3.0% or 3.3%) by weight carbidopa.

The formulation of any of the above embodiments may include about 25 toabout 40% (e.g., about 32% to about 40%, about 32%, or about 36%) byweight of a component selected from the group consisting of arginine,meglumine, and a combination thereof. For example, the formulation mayinclude 32% arginine, 32% meglumine, 36% arginine, or 36% meglumine.

The formulation of any of the above embodiments may, after storage for1, 2, 3, 4, 6, 8, 10, 12, 15, 18, 20, or 24 hours; 1, 2, 3, 5, 7, 10,14, 21, 28, or 30 days; 1, 2, 3, 4, 6, 9, or 12 months; or 1, 1.5, 2,2.5, or 3 years, at 25° C., 2-8° C. or at −20° C., have less than about5 μg/ml, less than about 1.0 μg/ml, or less than about 0.1 μg/ml ofhydrazine, as determined by GCMS.

The formulation of any of the above embodiments may have less than about5% (e.g., less than 4%, 3%, 2%, 1%, 0.5%, 0.3%, 0.2%, 0.1% or 0.05%) byweight 3,4-dihydroxyphenyl-2-methylpropionic acid (Degradant RRT 1.4),relative to the amount of carbidopa, as determined by HPLC.

The formulation of any of the above embodiments may, after storage for1, 2, 3, 4, 6, 8, 10, 12, 15, 18, 20, or 24 hours; 1, 2, 3, 5, 7, 10,14, 21, 28, or 30 days; 1, 2, 3, 4, 6, 9, or 12 months; or 1, 1.5, 2,2.5, or 3 years at 25° C., have less than 5% (e.g., less than 4%, 3%,2%, 1% 0.75%, 0.6%, 0.5%, 0.4%, 0.3%, 0.25%, 0.2%, 0.1%, 0.05%, or0.01%) by weight 3,4-dihydroxyphenyl-2-methylpropionic acid, asdetermined by HPLC. In other embodiments, the formulation may, afterstorage for 1, 2, 3, 4, 6, 8, 10, 12, 15, 18, 20, or 24 hours; 1, 2, 3,5, 7, 10, 14, 21, 28, or 30 days; 1, 2, 3, 4, 6, 9, or 12 months; or 1,1.5, 2, 2.5, or 3 years, at 2-8° C., have less than 0.5% (e.g., lessthan 0.3%, 0.2%, 0.1%, or 0.01%) by weight3,4-dihydroxyphenyl-2-methylpropionic acid, as determined by HPLC. Inother embodiments, the formulation may, after storage for 1, 2, 3, 4, 6,8, 10, 12, 15, 18, 20, or 24 hours; 1, 2, 3, 5, 7, 10, 14, 21, 28, or 30days; 1, 2, 3, 4, 6, 9, or 12 months; or 1, 1.5, 2, 2.5, or 3 years, at−20° C., have less than 0.2% (e.g., less than 0.15%, 0.1%, 0.05%, or0.01%) by weight 3,4-dihydroxyphenyl-2-methylpropionic acid, asdetermined by HPLC.

The formulation of any of the above embodiments may be in a formselected from the group consisting of liquid, gel, cream, solid, film,emulsion, suspension, solution, and aerosol (e.g., a liquidformulation).

In another aspect, the invention features a pharmaceutically acceptableliquid formulation including about 4% to about 8% (e.g., about 6%) byweight levodopa, about 0.1% to about 1.5% (e.g., about 0.6% to about1.4%, about 0.75%, or about 1.4%) by weight carbidopa, about 10% toabout 20% (e.g., about 15% to about 16%, about 15.2%, or about 15.6%) byweight arginine, and about 0.1% to about 1.5% (e.g., about 0.4 to about1%, about 0.4% to about 0.6%, or about 0.5%) by weight ascorbic acid ora salt thereof, e.g., where the formulation, after 1 day at 25° C., or30 days at 25° C. or after 180 days at 25° C., has less than about 20,2, 5, 1.0, 0.75, 0.5, 0.2, 0.1, or 0.05 μg/ml hydrazine, as determinedby GCMS. The formulation may further include about 0.1% to about 0.7%(e.g., about 0.4% or about 0.5%) by weight of L-cysteine or NAC. In aparticular embodiment, the formulation includes (a) about 0.4% to about0.6% or about 0.4 to about 1% by weight ascorbic acid or a salt thereofand (b) about 0.1% to about 0.7% by weight of L-cysteine or NAC. In thisaspect, the formulation may further include about 0.1% to about 0.5%(e.g., about 0.3%) by weight Tween-80.

In another aspect, the invention features a pharmaceutically acceptableliquid formulation including about 8% to about 16% (e.g., about 12% toabout 15%, about 12%, or about 13.2%) by weight levodopa, about 1% toabout 4% (e.g., about 3.0% or about 3.3%) by weight carbidopa, about 20%to about 42% (e.g., about 32% to about 42%, about 32%, or about 36%) byweight of a component selected from the group consisting of arginine,meglumine, and a combination thereof, and about 0.1% to about 1.5%(e.g., about 1.0% to about 1.4%, about 1.2%, or about 1.3%) by weightascorbic acid or a salt thereof (e.g., sodium ascorbate), e.g., wherethe formulation, after 1, 2, 3, 4, 6, 8, 10, 12, 15, 18, 20, or 24hours; 1, 2, 3, 5, 7, 10, 14, 21, 28, or 30 days; 1, 2, 3, 4, 6, 9, or12 months; or 1, 1.5, 2, 2.5, or 3 years at 25° C., has less than about20, 5, 2, 1.0, 0.75, 0.5, 0.2, 0.1, or 0.05 μg/ml hydrazine asdetermined by GCMS. The formulation may further include about 0.1% toabout 1% (e.g., about 0.1% to about 0.5%, about 0.3%, or about 0.5%) ofL-cysteine or salt thereof (e.g., cysteine HCl) or NAC. In a particularembodiment, the formulation includes about 0.1% to about 0.5% ofL-cysteine or NAC, and about 1.0 to about 1.4% by weight ascorbic acidor salt thereof.

In some embodiments, a pharmaceutically acceptable liquid formulation ofthe invention includes less than about 15%, less than about 12.5%, lessthan about 10%, less than about 7.5%, less than about 5%, less thanabout 2.5%, less than about 1%, or less than about 0.1% by weight (e.g.,about 0.1% to about 1% by weight or less) of a degradant relative to theamount of carbidopa or an ester thereof, as determined by HPLC. In someembodiments the degradant is, for example, a compound represented by thegeneral formula (I):

or a pharmaceutically acceptable salt thereof, wherein R¹, R², and R³are each independently selected from the group consisting of hydrogen,—R⁷(O)(OH)₂, and —R⁵—O—R⁷(O)(OH)₂; R⁵ is a C₁-C₄-alkyl; R⁶ is hydrogenor a C₁-C₄-alkyl; and R⁷ is O, P, F, or Cl. For example, in someembodiments, a pharmaceutically acceptable liquid formulation of theinvention includes less than about 15%, less than about 12.5%, less thanabout 10%, less than about 7.5%, less than about 5%, less than about2.5%, less than about 1%, or less than about 0.1% by weight of adegradant that is a compound of formula (I), relative to the amount ofcarbidopa, as determined by HPLC. In some embodiments, the degradantthat is a compound of formula (I) is3,4-dihydroxyphenyl-2-methylpropionic acid or a pharmaceuticallyacceptable salt thereof. In some embodiments, a pharmaceuticallyacceptable liquid formulation of the invention includes less than about15%, less than about 12.5%, less than about 10%, less than about 7.5%,less than about 5%, less than about 2.5%, less than about 1%, or lessthan about 0.1% by weight (e.g., about 0.1% to about 1% by weight orless) of 3,4-dihydroxyphenyl-2-methylpropionic acid or apharmaceutically acceptable salt thereof, relative to the amount ofcarbidopa, as determined by HPLC.

In another aspect, the invention features a method for treating adisease (e.g., Parkinson's disease) or condition associated with loss ofdopamine or dopaminergic neurons in a patient (e.g., a human ornon-human animal, such as a mammal). The method includes administeringto the patient an amount of the pharmaceutically acceptable formulationof any of the above aspects in an amount effective to the disease orcondition in the patient. The method may include substantiallycontinuous administration of the formulation. For example, providedherein is a method for treating Parkinson's disease comprisingsubstantially continuously administering to a patient in need thereof aliquid composition comprising levodopa or ester thereof, and orcarbidopa or ester thereof, and one, two or more antioxidants, such asascorbic acid or a salt thereof, such as a disclosed composition, whichadministers less than about 42 μg/day of hydrazine to the patient.

In another aspect, the invention features a method of reducing theimpurities in a formulation containing levodopa and carbidopa. Themethod includes (a) mixing together all powders (levodopa and/orcarbidopa, L-arginine and/or meglumine, antioxidants); (b) adding themixture of step (a) into pre-heated water or bringing the mixture to atemperature and for a time sufficient to dissolve the powders to form asolution; (c) cooling the solution to room temperature. The method mayfurther include (d) adding additional water, antioxidants, and/orTween-80, to the solution of step (c). In certain embodiments, the waterin step (b) includes antioxidants prior to the mixing. The method may beused to produce a formulation of any one of the above aspects.

In another aspect, the invention features a kit including one, two, ormore containers having a formulation of any one of the above aspects,where the formulation is present in an amount sufficient to treat apatient for a disease (e.g., Parkinson's disease) or condition resultingfrom decreased dopamine for at least 1, 2, 3, 4, or 5 days; 1, 2, 3 or 4weeks; 1 to 12 (e.g., 1 to 2, 3, 4, 6, or 9) months; or 1, 1.5, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20 years, or more. In certain embodiments, theformulation is present in separate dosages.

In certain embodiments of any of the above aspects, the formulation,method, or kit further comprises or comprises the use, of a secondagent. The second agent may be a catechol-O-methyl transferase (COMT)inhibitor, such as tolcapone or entacapone, or a pharmaceuticallyacceptable salt thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph depicting the main impurity (“Degradant”) at retentiontime of about 14.5±0.2 min.

FIGS. 2A and 2B are graphs depicting typical MS spectrum in negative(FIG. 2A) and positive (FIG. 2B) mode of collected main impurity peakfrom a formulation sample.

FIGS. 3A and 3B are graphs depicting typical MS/MS daughter scan (ionM/Z=179) spectrum (FIG. 3A) and parent (ion M/Z=105) spectrum (FIG. 3B)in collected main impurity peak from a formulation sample.

DETAILED DESCRIPTION

Provided herein are, in an embodiment, carbidopa formulations andlevodopa/carbidopa (LD/CD) formulations that can include, for example,one, two, or more antioxidant agents or o-quinone scavenger agents(e.g., ascorbic acid and L-cysteine, or ascorbic acid andN-acetylcysteine (NAC)). Such formulations, particularly those includingtwo antioxidants (e.g., one of them being L-cysteine or NAC) andparticularly when LD is present, can result in substantially lowerlevels of hydrazine, as compared to formulations with fewerantioxidants.

Disclosed formulations can include at least such two agents (e.g.,ascorbic acid and L-cysteine, or ascorbic acid and NAC, or sodiumascorbate and L-cysteine, or sodium ascorbate and NAC). Suchformulations can, e.g., reduce formation of undesired degradationproducts and/or provide substantially stable formulations. For example,provided herein are formulations that include carbidopa, having lessthan 10 μg/ml, 5 μg/ml, 1 μg/ml, 0.1 μg/ml or less than 0.5 μg/mlhydrazine, as determined by GCMS and/or less than 5% or less than 1% byweight 3,4-dihydrooxyphenyl-2-methylpropionic acid (relative to theamount of carbidopa), as determined by HPLC.

By “substantially continuous” administration is meant that a dose of theformulation being administered not administered as a bolus, e.g., a pilltaken orally or a bolus injection. For example substantially continuousadministration can involve administration of a dosage at over a periodof at least 10 minutes, 30 minutes, 1 hour, 2 hours, 4, hours, 6 hours,8 hours, 12 hours, 15 hours, 18 hours, 21 hours, or 24 hours toadminister a single dose. Substantially continuous administration can beachieved using a transdermal patch or a pump device that continuouslyadministers the formulation to a patient over time.

The term “pharmaceutically acceptable carrier” or “pharmaceuticallyacceptable excipient” as used herein refers to any and all solvents,dispersion media, preservatives, antioxidants, coatings, isotonic andabsorption delaying agents, and the like, that are compatible withpharmaceutical administration. The use of such media and agents forpharmaceutically active substances is well known in the art. Thecompositions can also contain other active compounds providingsupplemental, additional, or enhanced therapeutic functions.

Pharmaceutically or pharmacologically “acceptable” include formulations,molecular entities, and compositions that do not produce an adverse,allergic or other untoward reaction when administered to an animal, or ahuman, as appropriate. For human administration, preparations shouldmeet sterility, pyrogenicity, general safety and purity standards asrequired by, e.g., the U.S. FDA, and the EMA.

The term “pharmaceutical composition” as used herein refers to acomposition or formulation comprising at least one active agent asdisclosed herein formulated together with one or more pharmaceuticallyacceptable carriers.

The term “physiologically acceptable pH” is understood to mean a pH of,e.g., a formulation or composition that facilitates administration ofthe formulation or composition to a patient without significant adverseeffects, e.g., a pH of about 4 to about 9.8 (for example, about 4±0.3 toabout 9.5±0.3).

“Ambient temperature” as understood by a person of skill in the art isrefers to a temperature of from about 10° C. to about 30° C. Inparticular embodiments, ambient temperature can be 25° C.

Percentages disclosed herein are by weight unless indicated otherwise.

Compositions and Formulations

Provided herein, in an embodiment, are formulations that includecarbidopa (and/or an ester thereof) and arginine or meglumine (or amixture thereof). The formulation can optionally include levodopa(and/or an ester thereof); one, two, or more o-quinone scavenger agents;and an antioxidant. Disclosed formulations can further include asurfactant (e.g., polysorbate 20, 40, 60, and 80, or a combinationthereof).

In an embodiment, disclosed formulations can include about 0.1% to about10% carbidopa, e.g., about 0.5% to about 8%, about 0.6% to about 5%,about 0.1% to about 1%, about 1% to about 2%, particularly about 0.75%,about 1.4%, or about 4%. For example, a disclosed formulation caninclude about 1% to about 3% by weight, about 2.5% to about 3.5% byweight, about 0.6% to about 4% by weight, or about 1.2% to about 4% byweight carbidopa. In certain embodiments, disclosed compositions includeabout 0.01% to about 6% by weight carbidopa, about 0.1% to about 6% byweight carbidopa, or about 1% about to 4% by weight carbidopa, e.g.,about 0.6% to 4% or about 1.2% to 3% or 4% by weight carbidopa.

Disclosed formulations include arginine and/or meglumine (or a saltthereof and/or a combination thereof). For example, a disclosedformulation can include about 0.1% to about 42%, e.g., about 1% to about10%, about 12% to about 18%, about 0.1% to about 40%, about 2% to about7%, about 3.2%, about 3.4%, about 3.6%, about 3.7%, or about 4.6%arginine and/or meglumine. In other embodiments, disclosed formulationsinclude about 10% to about 20%, about 10% to about 25%, 12% to about18%, about 12.8%, about 14.8%, about 15.2%, about 15.5%, or about 18.5%arginine and/or meglumine (or a combination thereof). In certainembodiments, arginine, meglumine, a salt thereof, or a combinationthereof are present at about 25% to about 40%, about 30% to about 38%,about 32% or about 36%.

In certain embodiments, disclosed formulations can also include aglucose amine which can, for example, replace some or all of thearginine present in the formulations.

Disclosed formulations described herein can optionally include levodopa.For example, in certain embodiments disclosed formulations include about1% to about 20% levodopa, e.g., about 2% to about 8%, about 4% to about7%, about 5%, or about 6%. In other embodiments, the formulationsinclude about 8% to about 20%, about 8% to about 16%, about 10% to about14%, about 11% to about 14%, about 12%, or about 13.2%. A disclosedformulation can have a molar ratio of carbidopa to arginine (ormeglumine) of about 1:1 to about 1:25 or to about 1:35.

Disclosed formulations can include one, two, or more anti-oxidants oro-quinone scavenger agents. For example, a disclosed formulation caninclude one, two, or more of an agent each independently selected fromthe group consisting of ascorbic acid or a salt thereof (e.g., sodiumascorbate, calcium ascorbate, potassium ascorbate, ascorbyl palmitate,and ascorbyl stearate, particularly sodium ascorbate), and cysteine or acysteine derivative (e.g., L-cysteine, N-acetylcysteine (NAC),glutathione, diacetylcystine, S-methyl-N-acetylcysteine amide, acetylderivatives of S-methyl-N-acetylcysteine methylhydrazide,S-methylcysteine morpholineamide, and S-methyl-N-acetylcysteinemorpholineamide, or a salt thereof). For example, a disclosedformulations can include an ascorbic acid, or salt thereof, and acysteine derivative, such as NAC.

Disclosed formulations can include other antioxidants, such asdi-tert-butyl methyl phenols, tert-butyl-methoxyphenols, polyphenols,tocopherols, and ubiquinones (e.g., caffeic acid).

Disclosed formulations can also include a tyrosinase inhibitor.Exemplary tyrosinase inhibitors include captopril, methimazole,quercetin, arbutin, aloesin, N-acetylglucoseamine, retinoic acid,α-tocopheryl ferulate, MAP (Mg ascorbyl phosphate), substrate analogues(e.g., sodium benzoate, L-phenylalanine), Cu⁺⁺ chelators, for example,Na_(e)-EDTA, Na_(e)-EDTA-Ca, DMSA (succimer), DPA (D-penicillamine),trientine-HCl, dimercaprol, clioquinol, sodium thiosulfate, TETA, TEPA,curcumin, neocuproine, tannin, and cuprizone.

Disclosed formulations can include ascorbic acid or salt thereof (e.g.,sodium ascorbate). For example, disclosed formulations can include 0.1%to about 10% or more ascorbic acid (or salt thereof), or about 0.1% toabout 2%, e.g., about 0.2% to about 1.5%, about 0.2% to about 2.0%,about 0.2% to about 2.5%, about 0.3% to about 1.2%, e.g., about 0.4%,about 0.5%, about 0.75%, about 0.85%, or about 1.0% by weight. Forexample, a disclosed formulation can include about 0.8% to about 1.3% orabout 1% to about 2.5% by weight ascorbic acid or salt thereof. In aparticular embodiment, a disclosed formulation can include 0.5% to about0.85%, or, e.g., about 0.5%, about 0.75%, about 0.85%, about 1.0%, about1.2%, or about 1.3% by weight sodium ascorbate or ascorbic acid.

In particular embodiments, disclosed formulations can include abisulfite, e.g., sodium bisulfite or other sulfite salts, e.g., sodiumhydrogen sulfite or sodium metabisulfite.

In an embodiment, disclosed formulations can include for example, NAC,L-cysteine, diacetylcystine, and/or glutathione. In particularembodiments, the formulations includes about 0.001% to about 5%, about0.01% to about 5%, or about 0.1% to about 5%, about 0.001% to about 1%,or about 0.01% to about 1%, or about 0.1% to about 1% by weight of acompound selected from the group consisting of NAC, L-cysteine,diacetylcystine, and/or glutathione. For example, a disclosedformulations can include about 0.01% to about 5%, e.g., about 0.05% toabout 1%, 0.1% to about 0.6%, about 0.1%, about 0.2%, about 0.3%, about0.4%, or about 0.5% of NAC and/or L-cysteine. In a particularembodiment, a disclosed formulation includes about 0.4% or 0.5% NAC. Inanother particular embodiment, a disclosed formulation includes about0.3%, about 0.4%, or about 0.5% L-cysteine.

For example, a disclosed formulations can include ascorbic acid (or asalt thereof) and a cysteine derivative, e.g., L-cysteine and/or NAC. Inan exemplary embodiment, a disclosed formulation includes 0.1% to about10% ascorbic acid (or salt thereof) and 0.001% to about 5% or about0.001% to about 1% by weight L-cysteine and/or NAC and/ordiacetylcystine and/or glutathione. In particular embodiments, thecomposition includes ascorbic acid and L-cysteine, sodium ascorbate andNAC, ascorbic acid and NAC, sodium ascorbate and L-cysteine, ascorbicacid and diacetylcystine, sodium ascorbate and diacetylcystine, ascorbicacid and glutathione, or sodium ascorbate and glutathione.

Contemplated formulations are liquid, and can include a surfactant. Forexample, polysorbate 20, 40, 60, or 80 may be present in a disclosedformulations at about e.g., about 0.01% to about 5%, about 0.1% to about0.5%, e.g., about 0.3%. In particular embodiments, polysorbate 80 ispresent at about 0.3%.

Such formulations or solutions can have a pH that is pharmaceuticallyacceptable for subcutaneous administration, e.g., a pH of about 8 toabout 10, for example, about 9.1 to about 9.8, e.g., 9.2 to 9.6 at 25°C.

Kits, Devices, and Articles

Contemplated herein, in part, is a patch suitable for transdermal orsubcutaneous administration of an active agent in a formulation asdisclosed herein, for example, including levodopa and carbidopa, andarginine. Such patches can have one or more compartments that can havethe same or different formulations, for example, one compartment canhave a disclosed formulation and another a different disclosedformulation, or a different active formulation. A dermal patch refers toany device that is capable of delivering one or more of the activeagents forming a disclosed formulation through the skin or mucousmembrane into the a patient.

In some embodiments, disclosed liquid formulations (e.g., comprisingcarbidopa, arginine, and optionally levodopa), can be provided in, e.g.,a pre-filled cartridge or vial suitable for use by a patient orphysician. For example, provided herein is a kit comprising a prefilledcartridge wherein a disclosed liquid formulation is disposed within thecartridge (e.g., a pre-filled cartridge having a single dose or a dosesuitable for a single or multiple administration to a patient of adisclosed formulation and optionally instructions for use). For example,provided herein is a container, vial, pre-filled syringe or the likethat can include about 1-10 ml of a disclosed formulation. For example,a contemplated kit can include one, two, or more pre-filled vial,container or syringe having an amount of a disclosed liquid formulationsuitable for filling a syringe pump or patch pump, e.g., a vial,container, or syringe having about 1-6 ml, 2-5 ml, 1-2 ml, or 4-10 ml ofa disclosed formulation.

The invention also contemplates kits that include formulations of theinvention that take advantage of their increased stability. These kitscan include a supply of a formulation of the invention sufficient for atleast 1, 2, 3, 4, or 5 days; 1, 2, 3 or 4 weeks; 1, 2, 3, 4, 6, or 9months; or 1 or 1.5 years of administration to a patient, which can bepackaged, for example, into suitable dosage (e.g., unit dosage)formulations. These kits can optionally include instructions for theiruse. A kit for daily use for example, can include one, two or morecontainers or vials of a disclosed formulation, an infusion set, and adisposable patient delivery units (e.g. syringe).

Preparation of Compositions

Disclosed formulations or compositions can be prepared by mixingarginine and/or meglumine in amounts as disclosed above with levodopaand/or carbidopa, and optionally anti-oxidant(s), e.g., to form a powdermixture. Water can be added to the mixture to form a suspension. Thewater can be pre-heated or the suspension can be heated at a temperatureand for a time sufficient to dissolve the mixture, e.g., to about 40° C.to about 100° C., or to about 40° C. to 80° C., or to about 60° C. to90° C., e.g., 65±5° C. or 72±5° C. or 73±3° C., e.g., by addingpre-heated water and/or by placing the mixture in a hot (e.g., 65±5° C.or 72±5° C. or 73±3° C.) water bath (e.g., for up to about 10 minutes,for about 3 minutes, for about 5 minutes, or for about 10, 20, 30, 40,50, 60 minutes, or more) to form a solution, with optional stirring.This is followed by cooling the solution to form the composition. N₂ canbe provided the head space of the container. For example, the mixturecan then be removed from the hot water bath and cooled to roomtemperature, and adding, e.g., immediately thereafter, an optionalanti-oxidant(s) under N₂ atmosphere and subsequent stirring. Apreparation such as that above, e.g., where levodopa and/or carbidopa,and arginine are mixed together as powders first, and a suspensionformed with water and then heated can result in a more stable solution,as compared to a preparation that includes a stepwise preparation ofindividual water suspensions of ingredients and later combination.Specific methods of preparation are described in Example 1 below.

Disclosed formulations can be sterilized, e.g., using 0.2 μm filterssuch as filters with nylon or PVDF membranes. In another embodiment, thepreparation of disclosed formulations has fewer undesirable by-productswhen pre-heated water is added as disclosed above, as compared to aformulation prepared without the addition of pre-heated water. Inanother embodiment, the levodopa and/or carbidopa cannot dissolve unlessthe preparation procedure disclosed is used. Such disclosed preparationsas above can provide a more stable formulation as compared to aformulation prepared without adding hot water or heating. Disclosedpreparations can provide a more stable formulation as compared to aformulation prepared without adding antioxidants prior to heating.

Methods of Treatment

In another aspect, the present invention contemplates a method fortreatment of a disease or disorder, such as a neurological disorder(e.g., a disorder associated with reduced dopamine or loss ofdopaminergic neurons) or a movement disorder, comprising administering aformulations described herein. The method can include substantiallycontinuously administering the formulation.

Also provided herein are methods of treating a neurological disorder(e.g., a disorder associated with reduced dopamine or death ofdopaminergic neurons) or a movement disorder that include substantiallycontinuous administration of a formulation described herein.

Without limiting, the pharmaceuticals compositions as described abovecan be used for treating a neurological disorder (e.g., a disorderassociated with reduced dopamine or death of dopaminergic neurons) or amovement disorder that include acute and immediate administration suchas inhalation or injection.

In some embodiments, compositions comprising levodopa (e.g. a disclosedliquid composition) may be administered at a rate of about 0.16ml/hour/site to about 0.24 ml/hour/site, or, e.g., about 0.01 ml/hour toabout 0.4 ml/hour/site. Such rates may be constant throughout the dayand night or varied according to patient's need, for example, mayreflect a patient resting or sleeping schedule and waking or higheractivity level schedule. For example, liquid compositions such as thosedisclosed herein (e.g., including levodopa) may be administered at arate of about 0.32 ml/hour/site in the morning (e.g., for about 2-4hours before waking), about 0.24 ml/hour/site during the daytime oractivity time, (e.g., for about 10 to about 12 hours), and/or about 0.08ml/hour/site at rest or at night. In another embodiment, liquidcomposition such as those disclosed herein may be administered, e.g.,intraduodenally, at a rate of about 1.0 ml/hour during the daytime oractivity time (e.g., for about 2-3 hours before waking and for about 10to about 12 hours thereafter), and 0 to about 0.5 ml/hour at rest or atnight. In another embodiment, liquid compositions such as disclosedherein (e.g., comprising levodopa and arginine), may be administered ata rate of about 1.25 ml/hour (e.g., about 1.25±0.5 ml/hour during thedaytime or activity time (e.g., for about 2-3 hours before or afterwaking and for about 10 to about 14 hours thereafter) and 0 to about 0.5ml/hour (e.g. about 0.5±0.25 ml/hour) at rest or night. In furtherembodiments, such compositions may be administered at a rate of about0.1 to about 1000 μl/hour/site; or at a volume of about 2 to about 10ml/24 hour/site, preferably about 4 to about 6 ml/24 hour/site; or at adose of about 80 to about 800 mg levodopa/day and about 20 to about 200mg carbidopa/day; or at a rate of about 240 to about 360 mg levodopa andabout 60 to about 90 mg carbidopa/day/site.

Contemplated administration, following the disclosed methods, typicallycan be carried out over a defined time period (usually weeks, months, oryears). Administration can be effected by any appropriate routeincluding, but not limited to, subcutaneous, oral routes, intravenousroutes, intramuscular routes, intradermal routes, subcutaneously,intratracheally, intrathecally, intraduodenally, transdermally,inhalation, and/or direct absorption through mucous membrane tissues.

The disease or disorder characterized by reduced levels of dopamine in apatient contemplated herein are neurological or movement disordersincluding restless leg syndrome, Parkinson's disease, secondaryparkinsonism, Huntington's disease, Shy-Drager syndrome, and conditionsresulting from brain injury including carbon monoxide or manganeseintoxication. Methods for treating such disorders in a patient in needthereof are provided, for example, by administering (e.g.,subcutaneously) a disclosed formulation. In one embodiment, the diseaseto be treated is Parkinson's disease.

In an embodiment, substantially continuously administering using, e.g.,a liquid formulation can be via a pump for subcutaneous infusion(insulin pump) at an average rate of about 10-1000 μl/hour (e.g., 10-250μl/hour), about 300±100 μl/hour, or about 200±40 μl/hour continuouslyfor 24 hours; about 440±200 μl/hour or about 200±50 μl/hour continuouslyfor 16 hours (during waking hours) and at night (e.g., for 8 hours,about 0 to 80 μl/hour or 0 to 200 μl/hour or via a pump or a transdermalpatch. Substantially continuously administering the formulation in to apatient can be doubled or tripled by using more than one pump or site ofinfusion. In an embodiment, substantially continuously administeringusing, e.g., a liquid formulation can be at an average rate of about0.2-2 μl/hour, or about 1±0.5 μl/hour continuously for 24 hours; about1.0±0.5 μl/hour continuously for 16 hours (during waking hours) and atnight (e.g., for 8 hours, about 0 to 0.5 μl/hour via a pump ortransdermal patch, or combination of delivery devices that are suitablefor, e.g., subcutaneous, intravenous, intrathecal, and/or via theduodenum).

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention inany way.

EXAMPLES Example 1: Formulation Preparation Procedure

Levodopa (LD) and carbidopa (CD) formulations can be prepared asfollows.

Method #1 (L-Arg Solution):

L-Arg and Na-Bis (Na-bisulfite) were dissolved in water. The solutionwas added to the LD and CD powders. The mixture was heated with stirringfor 13 min at 75° C. until fully dissolved. The LD/CD solution was keptat room temperature (RT) for 10 min to cool down.

Method #2 (all Powders Together):

All powders (LD, CD, and L-Arg) were weighed, and water with Na-Bis wasadded. The suspension was heated with stirring for 13 min at 75° C.until fully dissolved. The LD/CD solution was kept at RT for 10 min tocool down.

Method #3 (Same as #2 without Na-Bis Pre-Heating):

All powders (LD, CD, and L-Arg) were weighed together and water wasadded. The suspension was heated with stirring for 13 min at 75° C.until fully dissolved. The LD/CD solution was kept at RT for 10 min tocool down.

Method #4 (Preparation in Steps):

LD and the respective amount of L-Arg were weighed; water and Na-Bissolution were added. The suspension was heated for 7 min at 75° C. untilfully dissolved, followed by 7 min at RT. CD and the respective amountof L-Arg were weighed and added to the LD/Arg solution at 60° C. untilfully dissolved. Finally, extra L-Arg was added.

Method #5 (Same as #4 without Na-Bis Pre-Heating):

LD and the respective amount of L-Arg were weighed; water was added. Thesuspension was heated for 7 min at 75° C. until fully dissolved followedby 7 min at RT. CD and the respective amount of L-Arg were weighed andadded to the LD/Arg solution at 60° C. until fully dissolved. Finally,extra L-Arg was added.

After cooling down, all formulations from all methods were divided in to3 vials, and water, Na-Bis solution, or Na-Bis-Arg solution was added toeach vial.

Example 2: Identification of the Main Degradant in FormulationsContaining Carbidopa

Liquid formulations with levodopa, carbidopa, and arginine were preparedusing the procedure outlined in Example 1, and HPLC analysis wasperformed according to APH stability-indicating analytical method forcarbidopa levodopa formulations with Agilent 1100 system.

The HPLC system used herein includes the following componentsmanufactured by Agilent: pump system (model G1311A), diode arraydetector (model G1315B), autosampler (model G1329A), degasser (modelG1379A), thermostat (model G1330B), thermostatted column compartment(model G1316A). The column employed was a new Synergi 4μ Fusion-RP 80A,250×4.6 mm (Phenomenex®).

HPLC working conditions:

-   Wavelength: 280 nm-   Flow rate: 1.0 ml/min-   Inj. volume: 10 μl-   Column temperature: 30° C.-   Thermostat temperature: 4° C.-   Stop time: 27 min-   Pressure: 105 bar

Mobile Phase Preparation:

-   Solvent A: Acetonitrile-   Solvent B: 20 mM potassium dihydrogen phosphate, pH=2.4    The mobile phase B was prepared by weighing 2.72 g/L of potassium    dihydrogen phosphate. pH was adjusted to 2.4 by addition of H₃PO₄.

Gradient:

Time Solvent A Solvent B Flow 0 5 95 1.0 5 5 95 1.0 15 60 40 1.0 20 6040 1.0 20.01 5 95 1.2 27 5 95 1.2

Diluent:

-   0.1M HCl/MeOH 9:1-   (8.3 ml HCl 37% to 1 L)-->0.1 M HCl-   STD LDOPA=100.00 mg/100 ml-   STD CDOPA=25.00 mg/100 ml

Calibration Curve:

STD stock sol. Volumetric Final concentr. 1000/250 ppm bottle, ml LD/CDppm NA 10 1000/250 5000 from stock 10  500/125 5000 from 500/125 10  250/62.5 1000 from stock 10 100/25 5000 from 100/25 10   50/12.5 1000from 100/25 10   10/2.5

One ml of the sample (levodopa/carbidopa formulation) was transferred toa 25 ml amber volumetric glass flask and filled to volume with diluent(0.1 M HCl/MeOH 9/1). The sample was degraded with hydrogen peroxide.

The impurity was observed at retention time of about 14.5±0.2 min (FIG.1). To ensure that the peak observed is actually the compound ofinterest, the main degradant peak was collected from analytical HPLC,evaporated under nitrogen stream and reconstituted with diluent.Obtained samples were tested by HPLC/MS.

Initially, an MS scan analysis was applied (FIGS. 2A and 2B). Theunknown compound shows clear and intensive signal in negative mode andmuch nosier signal in positive mode. Therefore it was expected to bemore a proton donor than acceptor. The base peak in negative mode wasM/Z=195 Da that was suspected as (M−H). The mass difference between thision and peak M/Z=217 is 22 and that should be the sodium adduct. This isthe evidence of the presence of carboxyl or/and phenol groups. Due tothis fact, the molecular weight was proposed to be 196 Da.

The peak M/Z=197 (M+H+)+ was not found in positive mode, but the peakM/Z=197 (M−H2O)+ is observed. This is typical for oxygen containingmolecule.

The daughter and parent MS/MS was performed as well to define molecularstructure. Peaks observed in positive mode withM/Z=179,161,151,133,123,105 were found to be relative. They were definedas in-source fragment ions arising from the molecular ion with M/Z=197.The typical MS and MS/MS spectra are shown on figures (FIGS. 3A and 3B).

The chemical formula of the degradant compound is C₁₀H₁₂O₄; with amolecular structure given by 3-(3,4 dihydroxyphenyl)-2-methylpropanoicacid.

Example 3: Effect of Ascorbic Acid With or Without EDTA on LD/CDFormulation Stability

Liquid formulations were prepared by weighing all powders (LD, CD, EDTA,ascorbic acid, and L-Arg) and adding water pre-heated to 73±3° C. Thesuspension was put in a water bath at 73±3° C. and stirred for 10 minuntil fully dissolved. LD/CD solution was kept at RT for 10 min to cooldown. Solutions were divided into glass vials and kept at +25° C. and at−20° C. for the indicated period of time. Prior to analyses, frozenvials were placed at RT until fully thawed. Formulations were then mixedand subjected to stability analyses. The effect of ascorbic acid with orwithout EDTA on stability of LD/CD formulations was measured by HPLC.The levels of the degradant presented in Tables A and B indicate thelevel of stability of LD/CD formulations.

TABLE A L-Arginine Ca-EDTA- Ascorbic t = 0 LD/CD (%) Na₂ (%) acid (%)Degradant Total (%) 6/1.4 14.80 0.2 1.0 0.13 0.76 6/1.4 14.80 0 1.0 0.080.44

TABLE B L-Arginine Ca-EDTA-Na₂ Ascorbic acid t = 0 t = 1 w (25°) LD/CD(%) (%) (%) Degradant Total (%) Degradant Total (%) 6.0/1.4 14.80 0.21.0 0.054 0.64 0.16 0.79 6.0/1.4 14.80 0 1.0 0.046 0.49 0.15 0.49Tables A and B indicate that EDTA did not have a significant effect onthe stability of LD/CD formulations.

Example 4: Effect of L-Cysteine on the Stability of Solutions ContainingCD

Liquid formulations were prepared by weighing all powders (LD, CD,L-cysteine, ascorbic acid, and L-Arg) and adding water pre-heated to73±3° C. The suspension was put in a water bath at 73±3° C. and stirredfor 10 min until fully dissolved. LD/CD solution was kept at RT for 10min to cool down. For CD formulations, CD, L-cysteine, and ascorbic acidwere weighed, and pre-heated water (60° C.) was added. Solutions weredivided into glass vials and kept at +25° C. and at −20° C. for theindicated period of time. Prior to analyses, frozen vials were placed atRT until fully thawed. Formulations were then mixed and subjected tostability analyses. The effect of L-cysteine on the stability ofcarbidopa formulations when stored at 25° C., either exposed to air ormaintained under anaerobic conditions (N₂), was analyzed using HPLC. Thelevels of the degradant presented in Table C point toward the level ofstability of carbidopa formulations.

TABLE C Ascorbic L- Degradant L- acid Cysteine t = 5 t = 5 LD/CDarginine (%) (%) t = 0 days weeks 6/1.4 14.8 0.5 0.1 N₂ 0.08 0.22 0.28O₂ 0.59 0.77 0.5 0.2 N₂ 0.08 0.20 0.25 O₂ 0.36 0.32 0.5 0.4 N₂ 0.00 0.140.14 O₂ 0.14 0.17 0/4  4.6 0.5 0.1 N₂ 0.05 0.16 O₂ 1.42 0.5 0.2 N₂ 0.050.16 O₂ 0.56 0.5 0.4 N₂ 0.04 0.15 O₂ 0.35

As Table C indicates, ascorbic acid with 0.1% L-cysteine was sufficientto inhibit degradant formation in formulations containing carbidopa(with or without levodopa) when kept under anaerobic conditions at 25°C. for at least 5 weeks. We can further deduce from Table C, thatL-cysteine reduced degradant formation under aerobic conditions at 25°C. in a dose-dependent manner.

In formulations containing carbidopa and 0.4% L-cysteine, degradantformation was inhibited during the preparation of the formulation. Theseformulations were stable for at least 5 weeks at 25° C. under bothaerobic and anaerobic conditions. Formulations containing carbidopa aremore stable when they also contain LD upon exposure to air.

Example 5: Effect of L-Cysteine on 6/1.4% Levodopa/Carbidopa FormulationStability

Liquid formulations were prepared as described in Example 3. The effectof L-Cysteine on the stability of 6/1.4% levodopa/carbidopa solutions at25° C. was analyzed using HPLC. The levels of the degradant presented intables D, E, F, and G point toward the level of stability of referencedformulations.

TABLE D LD/CD L-Arginine Ascorbic Acid L- Cysteine Degradant (%) (%) (%)(%) t = 0 6/1.4 14.8 0.5 0.2 0.02 0.75 0.0 0.16

TABLE E L- Ascorbic L- LD/CD arginine acid Cysteine Degradant (%) (%)(%) (%) t = 0 t = 5 d t = 5 wk 6/1.4 14.8 0.75 0 0.49 0.93 1.08 0.1 0.100.35 0.32 0.50 0.1 0.16 0.28 0.45 0.4 0.00 0.15 0.13

TABLE F L- Ascorbic L- LD/CD arginine acid Cysteine Degradant (%) (%)(%) (%) t = 0 t = 1 d t = 4 d 6/1.4 14.8 0.5 0.4 0.00 0.00 0.27 0.1 0.250.69 1.26 0.75 0.1 0.23 0.56 0.85 15.1 1.0 0.2 0.00 0.26 0.51

TABLE G LD/CD L-arginine Ascorbic acid L-Cysteine Degradant (%) (%) (%)(%) t = 0 t = 10 d t = 3 weeks t = 3 months 6/1.4 14.8 0.5 0.1 0.15 0.430.46 0.87 0.75 0.0 1.01 1.40 1.37 2.00

Results show that levodopa/carbidopa formulations were more stable withboth ascorbic acid and L-cysteine, as compared to ascorbic acid alone,suggesting that L-cysteine and ascorbic acid have a synergistic effectin preventing degradant formation. Other results showed that L-cysteinealone had no effect at all (data not shown). Furthermore, L-cysteineinhibited degradant formation during formulation preparation andmaintained the stability of the formulation for at least up to 5 weeksat 25° C., in a dose dependent manner. Increasing the amount of ascorbicacid reduces degradant formation, but this was significantly lessefficient than the combination of ascorbic acid with L-cysteine.

Example 6: Effect of Tween-80 and Na-Ascorbate on Levodopa/CarbidopaFormulation Stability

Liquid formulations were prepared by weighing all powders (LD, CD,L-Cysteine, ascorbic acid, Na-ascorbate and L-Arg) and adding waterpre-heated to 73±3° C. The suspension was put in a water bath at 73±3°C. and stirred for 10 min until fully dissolved. LD/CD solution was keptat RT for 10 min to cool down. Then, Tween-80 was added. Solutions weredivided in to glass vials and kept at +25° C. and at −20° C. for theindicated period of time. Prior to analyses, frozen vials were placed atRT until fully thawed. Formulations were then mixed and subjected tostability analyses. The effect of Tween-80 and Na-ascorbate on thestability of carbidopa/levodopa formulations was analyzed using HPLC.The levels of the degradant presented in table H point toward the levelof stability of carbidopa/levodopa formulations.

TABLE H LD:CD L-Arginine Asc. acid:L-Cys Tw-80 Degradant (%) (%) (%) (%)t = 0 t = 1 month 6.0:1.5 14.8  0.5:0.4 0 0.07 0.08 6.0:1.5 14.8 0.5:0.4 0.3 0.10 N/A 6.0:1.5 14.8  0.5:0.4 0.75 0.15 6.0:1.5 14.8 0.5:0.4 2.0 0.00 6.0:1.5 14.8 0.75:0.1 0 0.26 6.0:1.5 14.8 0.75:0.10.75 0.31 6.0:1.5 14.8 0.75:0.2 0 0.14 6.0:1.5 14.8 0.75:0.2 0.3 0.276.0:1.5 14.8 0.75:0.2 0.75 0.24 6.0:1.5 14.8 0.75:0.2 2.0 0.35 7.5:1.518.5 0.75:0.2 0 0.19 0.26 7.5:1.5 18.5 0.75:0.2 0.75 0.35 0.45 7.5:1.518.5 0.85*:0.2  0 0.23 0.20 7.5:1.5 18.5 0.85*:0.2  0.75 0.25 0.27*Sodium ascorbate

The results demonstrate that Tween-80 did not have an effect ondegradant formation. It is also shown that the effect of L-cysteine onthe stability of the formulations was dose dependent. Table H furthershows that the effect of Na-ascorbate and ascorbic acid on the stabilityof the formulations and degradant formation was similar.

Example 7: Effect of Ascorbic Acid With or Without L-Cysteine or NAC onLong Term Stability of Levodopa/Carbidopa Formulations

Liquid formulations were prepared by weighing all powders (LD, CD,arginine, L-Cysteine or NAC, and ascorbic acid or Na-ascorbate) and byadding water pre-heated to 73±3° C. The suspension was put in a waterbath at 73±3° C. and stirred until fully dissolved. LD/CD solution waskept at RT to cool down. Then Tween-80 was added. Solutions were dividedinto glass vials and kept at +25° C. and at −20° C. for the indicatedperiod of time. Prior to analyses, frozen vials were placed at RT untilfully thawed. Formulations were then mixed and subjected to stabilityanalyses. The effect of ascorbic acid with or without L-cysteine or NACon the stability of carbidopa/levodopa formulations was analyzed usingHPLC. The levels of the degradant presented in Table I indicate thelevel of stability of carbidopa/levodopa formulations.

TABLE I Degradant 2 × F − T* LD/CD Ascorbic (NAC) Tween- Arginine −20°C. 25° C. (ambient) (%) acid (%) (%) 80 (%) (%) T₀ 1 m 2 m 3 m 6 m 9 m12 m 16.5 m 1 m >3 m/>7 d  5/1.15 0.75 0 0 12.8 0.55 0.6 0.75 0.9 1.00.8 — 1.0 1.2 — 6/1.4 0.75 0 0 14.8 0.4 0.45 0.45 0.6 0.5 0.8 — 0.6 0.6— 6/1.4 0.5 0.4 0 14.8 0 0 — — — 0.25 0.15 — 0 — 6/1.4 0.5 0.4 0.3 15.50 — 0.1  0.2 0.2 0.2 — — — — 6/1.4 0.5 (0.5) 0.3 15.5 0 0 — — — — — —0.2 0  6/0.75 0.5 (0.5) 0.3 15.2 0 0.1 — — — — — — 0 0.2 *2 × F/T—atleast 2 freeze-thaw cycles after >3 m at −20° C., and >7 d at ambienttemp.

The results presented in Table I suggest that formulations containingboth ascorbic acid and L-cysteine or NAC are more stable thanformulations having only ascorbic acid, at a) T₀, i.e., immediatelyfollowing formulation preparation, b) for at least 9 months at −20° C.,and c) at for least 1 month at ambient temperature.

Example 8: Effect of Antioxidants on the Stability of CarbidopaFormulations

Liquid formulations with carbidopa and arginine were prepared asdescribed above. The effect of antioxidants on the stability ofcarbidopa formulations was analyzed using HPLC. The levels of thedegradant presented in Tables J and K point toward the level ofstability of carbidopa formulations.

TABLE J 1 2 3 4 7 weeks at 0.1% Na 0.075% Na 0.4% Asc. + 0.5% Asc. +(−20° C.) Bisulfite Bisulfite 0.2% L-Cys 0.1% L-Cys Methyl-Dopa 0.150.15 0.15 0.15 Unknown 2 0.18 0.17 0 0 Degradant 0.54 0.55 0 0.16 Sum ofall 1.14 1.15 0.44 0.65 Impurities

TABLE K Peak 14.3 min (degradant) t = 3 weeks t = 3 weeks t = 0 2-8° C.25° C. 2 4% CD - 3.7% L-Arg 0.30 1.12 ± 0.19 1.08 ± 0.07 0.075% sodiumbisulfite 3 4% CD - 4.62% L-Arg 0.06 0.15 ± 0.01 0.29 ± 0.07 0.4%ascorbic + 0.2% L-cysteine 4 4% CD - 4.62% L-Arg 0.11 0.44 ± 0.21 0.63 ±0.39 0.5% ascorbic + 0.1% L-cysteine

The results in Tables J and K suggest that formulations containingascorbic acid+L-cysteine were significantly more stable than theformulation containing Na-bisulfite (formulations 3 & 4 vs. formulations1 & 2). The same amount of impurities were measured with 0.075 and 0.1%Na-bisulfite, suggesting that the maximum possible protection withNa-bisulfite was attained.

In sum, the combination of ascorbic acid/L-cysteine is able to preventdegradant and other impurities formation, such as hydrazine (see otherexamples), while Na-bisulfite does not protect formulations containingcarbidopa to the same extent.

Example 9: Effect of Various Antioxidants and Different Concentrationsof Arginine on the Stability of Formulations Containing 4% CD

Liquid formulations with carbidopa and arginine (Table L) were preparedas described above. The effect various antioxidants and differentconcentrations of arginine on the stability of formulations containing4% CD, and stored under aerobic (air) or anaerobic (N₂) conditions, atambient (25° C.) or cold (2-8° C.) temperature was evaluated using HPLCanalysis. The levels of the degradant and total impurities as presentedin Tables M and N, respectively, point toward the level of stability ofcarbidopa formulations.

TABLE L 1 2 3 Carbidopa 4.0 4.0 4.0 L-Arginine 4.6 4.6 3.7 Ascorbic acid0.4 0 0 L-Cysteine 0.2 0 0 Na bisulfite (5%) 0 1.5 1.5 Total solutes 9.210.1 9.2 Water 90.8 89.9 90.8 L-Arg (mM) 265 265 212 CD (mM) 177 177 177Ratio L-Arg/CD 1.5 1.5 1.2 pH measured 8.67 8.87 8.62

TABLE M degradant: Peak area (%) T = 1 week T = 1 week 2-8° C. 25° C. t= 0 Air N2 Air 1 0.4% ascorbic + 0.2% cysteine 0.1 1.7 0.2 5.8 (CD:Arg1.0:1.5) 2 0.075% Bisulfite 0.2 1.9 1.2 6.2 (CD:Arg 1.0:1.5) 3 0.075%Bisulfite 0.25 2.4 1.2 8.3 (CD:Arg 1.0:1.2)

TABLE N Total degradation products: Peak area (%) T = 1 week T = 1 week2-8° C. 25° C. t = 0 Air N2 Air 1 0.4% ascorbic + 0.2% cysteine 0.1 2.51.1 7.2 (CD:Arg 1.0:1.5) 2 0.075% Bisulfite 0.2 2.6 2.1 7.7 (CD:Arg1.0:1.5) 3 0.075% Bisulfite 0.25 3.1 2.1 9.8 (CD:Arg 1.0:1.2)

The results as presented in Tables M and N indicate that formulationscontaining more arginine are more stable when exposed to air at 25° C.(formulations 2 vs. 3). Further, formulations containing Na-bisulfiteare less stable than the formulation containing ascorbic acid andL-cysteine (formulations 2 vs. 1, respectively) when stored undernitrogen (anaerobic conditions). N₂ provided significant protection fromdegradation and degradant formation. Formulations exposed to air aremore stable when kept refrigerated, as compared to room (ambient)temperature.

Example 10: Effect of Various Antioxidants on the Stability ofFormulations Containing 4% Carbidopa at 40° C.

Liquid formulations with carbidopa and arginine (Table 0) were preparedas described above. The effect various antioxidants on the stability offormulations containing 4% CD at 40° C. was evaluated using HPLCanalysis. The levels of the degradant and total impurities, presented intables P and Q respectively, indicate the level of stability ofcarbidopa formulations.

TABLE O 1 2 3 4 Carbidopa 4.0 4.0 4.0 4.0 L-Arginine 3.4 3.7 4.6 4.6N-MP 3.5 0 0 0 Ascorbic acid 0 0 0.4 0.5 Cysteine 0 0 0.2 0.1 Nabisulfite 0.1 0.075 0 0 Total solutes 12.9 9.2 9.2 9.2 Water 87.1 90.890.8 90.8 L-Arg (mM) 195 212 265 265 CD (mM) 177 177 177 177 RatioL-Arg/CD 1.1 1.2 1.5 1.5 pH measured 8.76 8.96 9.13 9.12

TABLE P Degradant t = 2 d/ t = 5 d/ t = 0 40° C. 40° C. 1 4% CD - 3.4%L-Arg + 0.27 ± 0.01 1.52 ± 0.56 2.60 ± 0.67 3.5% N-MP 0.1% sodiumbisulfite 2 4% CD - 3.7% L-Arg 0.30 ± 0.01 1.41 ± 0.54 2.95 ± 0.600.075% sodium bisulfite 3 4% CD - 4.62% L-Arg 0.06 ± 0.01 0.38 ± 0.141.26 ± 0.45 0.4% ascorbic + 0.2% L-cysteine 4 4% CD - 4.62% L-Arg 0.11 ±0.01 0.50 ± 0.23 1.97 ± 0.52 0.5% ascorbic + 0.1% L-cysteine

TABLE Q Total impurities t = 2 d/ t = 5 d/ t = 0 40° C. 40° C. 1 4% CD -3.4% L-Arg + 3.5% 0.61 2.00 ± 0.54 3.19 ± 0.65 N-MP 0.1% sodiumbisulfite 2 4% CD - 3.7% L-Arg 0.60 1.86 ± 0.56 3.61 ± 0.60 0.075%sodium bisulfite 3 4% CD - 4.62% L-Arg 0.29 0.92 ± 0.16 2.15 ± 0.59 0.4%ascorbic + 0.2% L-cysteine 4 4% CD - 4.62% L-Arg 0.34 1.17 ± 0.28 2.82 ±0.61 0.5% ascorbic + 0.1% L-cysteine

The results presented in Tables P and Q suggest that formulationscontaining Na-bisulfite are less stable than the formulations containingascorbic acid and L-cysteine (formulations 1 & 2 vs. 3 & 4), both duringpreparation and when stored at 40° C. Furthermore, there was a cysteinedose-response, i.e., the higher the concentration of L-cysteine, theless degradant was formed. No dose response was observed withNa-bisulfite, suggesting that the maximum possible protection may beattained with 0.075% Na-bisulfite.

Example 11: Effect of Ascorbic Acid Combined with Various Antioxidantson the Stability of Formulations Containing 4% Carbidopa

Liquid formulations with carbidopa and ascorbic acid with or withoutadditional antioxidants were prepared as described above. Thecombination effect between ascorbic acid and various antioxidants on thestability of formulations containing 4% CD at 25° C. was evaluated usingHPLC analysis. The levels of the degradant and total impuritiespresented in tables R and S respectively, point toward the level ofstability of carbidopa formulations.

TABLE R Degradant t = 3 d at t = 2 d at 4% CD t = 0 25° C./N₂ 25° C./O₂Without antioxidants 0.24 1.29 3.27 0.5% Asc 0.54 3.17 5.09 0.5% Asc +0.2% bisulfite 0.16 0.76 2.98 0.5% Asc + 0.2% cysteine 0.09 0.27 2.670.5% Asc + 0.2% NAC 0.14 0.83 3.64 0.75% bisulfite (CONTROL) 0.39 1.232.98

TABLE S Total impurities t = 3 d at t = 2 d at 4% CD t = 0 25° C./N₂ 25°C./O₂ Without antioxidants 1.04 1.88 4.01 0.5% Asc 1.07 3.96 6.40 0.5%Asc + 0.2% bisulfite 0.70 1.29 3.64 0.5% Asc + 0.2% cysteine 0.67 0.743.43 0.5% Asc + 0.2% NAC 0.72 1.36 4.49 0.75% bisulfite (CONTROL) 1.131.71 3.58

Results presented in Tables R and S show that ascorbic acid, 0.5%, wasinsufficient for the prevention of degradant formation in a formulationcontaining carbidopa. Furthermore, ascorbic acid requires anotherantioxidant in order to exert its maximum antioxidant activity, forexample ascorbic acid, 0.5%, and L-cysteine, NAC, or Na-bisulfiteinhibited carbidopa degradation in a synergistic manner. Formulationscontaining ascorbic acid and L-cysteine had the lowest amount ofdegradant after 3 days at 25° C.

The effect of Na-bisulfite on carbidopa degradation was similar to thatobtained with no antioxidants at all.

Example 12: Effect of Antioxidants on the Stability of 4% CDFormulations Stored at 25° C.

Liquid formulations with carbidopa and arginine (Table T) were preparedas described above. The effect of various antioxidants on the stabilityof formulations containing 4% carbidopa at 25° C. was evaluated usingHPLC analysis. The levels of the degradant and total impuritiespresented in Table W, point toward the level of stability of carbidopaformulations.

TABLE T 1 2 3 4 5 6 7 Carbidopa 4.0 4.0 4.0 4.0 4.0 4.0 4.0 L-Arginine4.6 4.6 4.6 4.6 4.6 4.6 4.6 Ascorbic acid 0.2 0.5 0.2 0.0 0.0 0.0 0.2Cysteine 0.0 0.2 0.2 0.2 0.2 0.0 0.0 Na bisulfite 0.0 0.0 0.0 0.0 0.10.1 0.1

TABLE W Degradant TOTAL Impurities t = 0 t = 1 wk t = 0 t = 1 wk 1 0.2%ascorbic acid 0.48 1.43 1.20 2.11 2 0.5% ascorbic acid/0.2% 0.12 0.170.81 0.72 cysteine 3 0.2% ascorbic acid/0.2% 0.12 0.21 0.85 0.94cysteine 5 0.2% cysteine 0.26 0.53 1.02 1.00 5 0.2% cysteine/0.1% 0.711.01 1.62 1.66 bisulfite 6 0.1% bisulfite 0.33 0.84 1.16 1.45 7 0.2%ascorbic acid/0.1% 0.26 0.70 0.99 1.49 bisulfite

Results presented in Table W suggest that ascorbic acid, bisulfite, orcysteine, each used alone, did not inhibit degradant formation.Combinations between bisulfite and cysteine or ascorbic acid did notinhibit degradant formation. There was a synergistic inhibitory effecton degradant formation between ascorbic acid and cysteine, but no suchsynergism between cysteine and bisulfite was observed. Such synergisticeffects can be seen between ascorbic acid and bisulfite (with higherascorbic acid concentrations). These results further suggest thatformulations containing the unique combination of ascorbic acid andcysteine may provide the best means for the inhibition of degradantformation.

Ascorbic acid, at 0.2%, was not sufficient for the prevention ofdegradant formation. With 0.2% cysteine, 0.5% ascorbic acid was moreeffective than 0.2% in reducing the total amount of impurities anddegradant formation, suggesting that at least 0.5% ascorbic acid with0.2% cysteine is desirable.

Example 13: Determination of the Level of Hydrazine in CarbidopaFormulations

The determination of hydrazine was carried out by derivatization usingAceton-d6. The hydrazine derivative was analyzed by gas chromatographymass spectrometry (GC/MS). The specific mass of hydrazine derivative wasmeasured in the selected ion monitoring mode (SIM-mode) according toSolvias standard operating procedures (SOP's).

Liquid formulations with carbidopa, levodopa, and arginine (Table X)were prepared as described above. The levels of hydrazine in thereferenced formulations were measured (Table Y).

TABLE X Formulation Composition (%) Formulation # 1 2 3 4 5 6 7 8 9 10Carbidopa 4   4   1.4 1.4 1.4 1.4 0.75 1.4 0.75 0.75 Levodopa — — 6 6 66 6 6 6 6 Arginine 4.6 4.6 14.8 15.5 15.5 15.5 15.2 15.5 15.2 15.2Ascorbic 0.4 0.5 0.75 0.5 0.5 0.5 0.5 0.5 0.5 0.5 acid L-Cysteine 0.20.1 — 0.4 0.4 — — 0.4 0.4 0.4 NAC — — — — — 0.5 0.5 — — — Tween 80 — — —— — — — — — 0.3 Sodium — — — — — — — — — — Ascorbate Formulation # 11 1213 14 15 16* 17** 18 19 20 Carbidopa 1.4 0.75 1.4 1.4 3 1.5 1.5 1.4 31.3 Levodopa 6 6 6 6 12 6 6 6 12 12 Arginine 15.5 15.2 15.5 15.5 32.315.5 15.5 15.5 32.3 29 Ascorbic 0.5 0.5 0.5 0.5 — 0.5 0.5 0.5 acidL-Cysteine 0.4 — — 0.4 0.3 0.4 0.4 0.4 NAC — 0.5 0.5 — — — — — 0.3 0.3Tween 80 0.3 0.3 0.3 0.3 — 0.3 0.3 0.3 Sodium — — — — 1.2 — — — 1.2 1.2Ascorbate *Sealed with N2 **Sealed with O₂

TABLE Y Time and temp prior to Storage in analysis (in- Hydrazine (ppm)vials use stability) 1 2 3 4 5 6 7 8 9 10 T = 0 at −20° C. 1 month 24 h25° C. 0.82 0.77 0.39 at −20° C. 48 h 0.73 0.71 0.43 24 h 37° C. 0.760.84 0.41 48 h 0.87 0.93 0.59 3 months <0.1 <0.1 at −20° C. 6 months 3free-thaw 0.1 0.1 at −20° C. cycles (25° C.) 0.1 9 months 0.1 at −20° C.1 year 24 h 25° C. <0.1 at −20° C. 37° C. <0.1 0.1 24 hr 37° C. 7 days<0.1 <0.1 <0.1 at 25° C. 24 h 37° C. 0.1 <0.1 1 month 25° C. Time andtemp prior to Storage in analysis (in- Hydrazine (ppm) vials usestability) 11 12 13 14 15 16 17 18 19 20 T = 0 <0.1 <0.1 <0.1 at −20° C.1 month 24 h 25° C. at −20° C. 48 h 24 h 37° C. 48 h 3 months 0.1 0.1 at−20° C. 6 months 3 free-thaw at −20° C. cycles (25° C.) 0.1 <0.1 0.1 0.19 months 0.1 0.1 at −20° C. 1 year 24 h 25° C. at −20° C. 37° C. 0.1<0.1 0.1 0.1 24 hr 37° C. <0.1 0.1 <0.1 7 days at 25° C. 24 h 37° C. 1month 0.1 0.3 0.1 25° C.

The results presented in Table Y clearly show that the levels ofhydrazine is at least 2 fold lower in levodopa formulations versusformulations without levodopa. Furthermore, formulations comprisingL-cysteine or NAC show at least 4-fold lower levels of hydrazinecompared to formulations without L-cysteine or NAC.

Example 14: Carbidopa/Levodopa Formulations

Based on the discoveries of combinations that have reduced degradant andhydrazine formation, we have developed new CD/LD formulations. Theseformulations are shown in Tables Z and AA below.

TABLE Z DS Ascorbic L- Tween- (%) LD CD Arginine Acid Cysteine NAC 80 pH1 6 1.4 15.5 0.5 0.4 — 0.3 9.4-9.6 2 6 1.4 15.5 0.5 — 0.5 0.3 9.4-9.6 36 0.75 15.2 0.5 0.4 — 0.3 9.4-9.6 4 6 0.75 15.2 0.5 — 0.5 0.3 9.4-9.6Margins 6 0.6-1.4 15-16 0.5 0.4 0.5 0.3 9.4-9.6

TABLE AA DS Sodium L- Cysteine- Tween- (%) LD CD Arginine MeglumineAscorbate Cysteine NAC HCl 80 pH 1 12 3 32 — 1.2 0.3 — — — 9.6-9.8 213.2 3.3 36 — 1.3 0.3 — — — 9.6-9.8 3 13.2 3.3 — 36 1.3 0.3 — — —9.6-9.8 4 12 3 — 32 1.2 — 0.3 — — 9.6-9.8 5 12 3 32 — 1.2 — 0.3 — —9.6-9.8 12-15 1.2-4 32-42 32-42 1.0-1.3 0.1-0.5 ≤2 9.6-9.8 * ** * Canreplace L-cysteine. ** Optionally added to stabilize the formulation.

Additional formulations that may be used in the context of thosedisclosed herein are provided in Table BB below. The formulations mayinclude additional components (e.g., any of those described herein).Tables CC and DD described further formulations that can be used in thecontext of those described herein.

TABLE BB Levodopa Carbidopa Amino Acid Other Conc. (%) Conc. (%) NameConc. (%) Name Conc. (%) 3 0 Lys 5.6 2.5 0 Lys 4.6 1.25 0 His 2.5 9.5 0Arg 15.9 4.8 1.4 Arg 11.0 4.8 1.4 Arg 12.1 4.8 1.4 Arg 12.7 5.4 1.5 Arg13.5 5.4 1.5 Arg 14.8 6 1.5 Arg 14.8 6 1.5 Arg 16.0 7 2 Arg 17.8 7 1.5Arg 14.1 Dextrose 5.0 8 1.5 Arg 15.7 Dextrose 5.0 10 1.5 Arg 19.2Dextrose 5.0 6 1.5 Arg 9.3 NaOH 4.6 5 0 Meglumine 10.8 8 1.5 Arg 15.7Meglumine 3.2 8 1.5 Arg 12.2 Meglumine 7.9 10 1.5 Arg 19.2 Meglumine 4.010 1.5 Arg 14.6 Meglumine 9.9 7 1.5 Arg 14.1 Meglumine 2.8 7 1.5 Arg10.7 Meglumine 6.9 6 1.5 Arg 13.5 Na-Asc 1 6 1.5 Arg 14.2 Na-Asc 1 6 1.5Arg 14.8 Na-Asc 1 6 1.5 Arg 16.0 Na-Asc 1 4.8 1.4 Arg 11.0 Na-Asc 1 4.81.4 Arg 11.6 Na-Asc 1 4.8 1.4 Arg 12.1 Na-Asc 1 4.8 1.4 Arg 12.7 Na-Asc1 6 1.5 Arg 14.8 Asc 1 6 1.5 Arg 15.8 Na-Asc 1 6 1.5 Arg 15.8 Asc 1 61.5 Arg 16.8 Na-Asc 1 6 1.5 Arg 16.8 Asc 1 5.4 1.5 Arg 12.3 Na-Asc 1 5.41.5 Arg 12.3 Asc 1 5.4 1.5 Arg 13.5 Na-Asc 1 5.4 1.5 Arg 13.5 Asc 1 5.41.5 Arg 14.8 Na-Asc 1 5.4 1.5 Arg 14.8 Asc 1 6 1.5 Arg 16.0 Asc 1 7 2Arg 17.8 Asc 1 7 2 Arg 17.8 Na-Asc 1 12 3 Arg 24.4 12 3 Arg 29.6 12 3Arg 32.1 7 0 Arg 7 0.5 Arg 7 1 Arg 7 1.5 Arg 7 2 Arg 6 0 Arg 13.5 6 0.5Arg 14.2 6 1 Arg 14.8 6 2 Arg 16.5 0 2

TABLE CC Property 1 2 3 API Concentration 2 & 4%   4%  0.6-20%CD:Arginine ratio 1:1.1-1.2   1:1.5 1:≥1 Exipients NMP 3.5% 0   0-15%concentra- Na-bisulfite 0.1% 0   0-0.2% tion Ascorbic Acid 0 0.75% 0-2%or more L-Cysteine 0  0.1% 0-0.5% or more or NAC Other anti- — —   0-2%oxidants Osmolality 650-750 300-400 200-1800 for SC No limits for ID pH8.2-8.6 8.6-9.1   8-9.8 Stability  25° C. 48-72 hrs ≥21 d 2 wks-≥2 yrs 4° C. Not stable ≥21 d 2 wks-≥2 yrs −20° C. ≥1 year ≥21 d 2 wks-≥2 yrsSC Infusion/24 hrs 2 ml 2 ml   0.1-20 ml

TABLE DD Property 4 5 6 API CD 0 or 1   1-2% 0-4% up Concen- or 2% to 6%tration LD   3-7%   5-7% 2.5-12% up to 14% Ratios LD to CD 6:1-6:33.5-4:1  1:1-10:0.5 Ratio or LD alone CD:Arginine 1:1.2 1:9-14  1:≥35 Ratio LD:Arginine 1:1.8-2.2   1:2-3.5 1:≥3.6 Ratio API:Argi- [1:1.21:2.3-2.5  1:≥1.8 nine CD:Arg + 1:2 Ratio LD:Arg] + 12.5% Arg ExepientsNMP 0 0 0 Na-bisulfite  0.075-0.15% 0   0-0.2% Ascorbic 0   0.75 0-2% ormore Acid Other anti- — — 0-2% oxidants Osmolal- 9/1% 1300-1500 —200-1800 for ity LD/CD SC 7/2%  950-1150 1200-1300 No limits LD/CD forID 6/1.5% 800-850 940-980 LD/CD 5/1.25% NT 790-830 LD/CD pH 8.5-9.59.2-9.6 9.1-9.8   Stability  25° C. ≥2 days ≥2 days ≥2 days  4° C. <2days ≥2 days ≥2 days −20° C. ≥2 days ≥2 days ≥2 days SC Infusion/24 hrs2 ml 2-6 ml 0.1-10 ml/site Intraduodenal/24 hrs — — 4-24 ml Intrathecal— — 1-1000 μl/day

EQUIVALENTS

While specific embodiments of the subject invention have been discussed,the above specification is illustrative and not restrictive. Manyvariations of the invention will become apparent to those skilled in theart upon review of this specification. The full scope of the inventionshould be determined by reference to the claims, along with their fullscope of equivalents, and the specification, along with such variations.

Unless otherwise indicated, all numbers expressing quantities ofingredients, reaction conditions, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless indicated to the contrary, thenumerical parameters set forth in this specification and attached claimsare approximations that may vary depending upon the desired propertiessought to be obtained by the present invention.

INCORPORATION BY REFERENCE

The entire contents of all patents, published patent applications,websites, and other references cited herein are hereby expresslyincorporated herein in their entireties by reference.

What is claimed is:
 1. A method for treating Parkinson's disease in apatient, the method comprising administering to the patient apharmaceutically acceptable formulation in an amount effective to treatthe Parkinson's disease in the patient, wherein the pharmaceuticallyacceptable formulation comprises: about 0.1% to about 10% by weightascorbic acid or a pharmaceutically acceptable salt thereof; about 0.01%to about 1% by weight of a component selected from the group consistingof: NAC, L-cysteine, and pharmaceutically acceptable salts thereof;about 2% to about 16% by weight levodopa or an ester thereof; and about0.6% to about 1.4% by weight carbidopa or an ester thereof, and whereinthe pharmaceutically acceptable formulation has less than about 10 μg/mlof hydrazine, as determined by a gas chromatography-mass spectrometry(GCMS) method.
 2. The method of claim 1, wherein the administering issubstantially continuous.
 3. The method of claim 1, wherein theformulation comprises less than about 1 μg/ml of hydrazine, asdetermined by a GCMS method.
 4. The method of claim 1, wherein theformulation comprises about 0.1 μg/ml to about 2 μg/ml of hydrazine, asdetermined by a GCMS method.
 5. The method of claim 1, wherein theformulation further comprises about 10% to about 40% by weight of acomponent selected from the group consisting of: a natural basic aminoacid, an amino sugar, and combinations thereof.
 6. The method of claim5, wherein the natural basic amino acid is arginine.
 7. The method ofclaim 5, wherein the amino sugar is meglumine.
 8. The method of claim 1,wherein the formulation comprises about 11% to about 15% by weightlevodopa.
 9. The method of claim 1, wherein the formulation comprisesabout 12% to about 14% by weight levodopa.
 10. The method of claim 1,wherein the formulation further comprises a surfactant.
 11. The methodof claim 10, wherein the formulation comprises about 0.01% to about 5%polysorbate
 80. 12. The method of claim 1, wherein the formulation after30 days at −20° C., has less than about 0.2% by weight3,4-dihydroxyphenyl-2-methylpropionic acid, as determined by HPLC. 13.The method of claim 1, wherein the formulation is a liquid formulation.14. The method of claim 1, wherein the pharmaceutically acceptable saltof ascorbic acid is sodium ascorbate.
 15. The method of claim 1, whereinthe formulation has less than about 5% or less than about 1% by weightof a degradant, relative to the amount of carbidopa or an ester thereof,as determined by HPLC.
 16. The method of claim 15, wherein the degradantis a compound represented by the general formula (I):

or a pharmaceutically acceptable salt thereof, wherein R¹, R², and R³are each independently selected from the group consisting of hydrogen,—R⁷(O)(OH)₂ and —R⁵—O—R⁷(O)(OH)₂; R⁵ is a C₁-C₄-alkyl; R⁶ is hydrogen ora C₁-C₄-alkyl; and R⁷ is O, P, F or Cl.
 17. The method of claim 16,wherein the formulation has less than about 5% by weight of the compoundrepresented by general formula (I), relative to the amount of carbidopaor an ester thereof, as determined by HPLC.
 18. The method of claim 16,wherein the formulation has less than about 1% by weight of the compoundrepresented by general formula (I), relative to the amount of carbidopaor an ester thereof, as determined by HPLC.
 19. The method of claim 16,wherein the compound represented by general formula (I) is3,4-dihydroxyphenyl-2-methylpropionic acid.
 20. A method for treatingParkinson's disease, the method comprising substantially continuouslysubcutaneously administering to a patient in need thereof a liquidcomposition comprising: about 0.1% to about 10% by weight ascorbic acidor a pharmaceutically acceptable salt thereof; about 0.01% to about 1%by weight of a component selected from the group consisting of: NAC,L-cysteine, and pharmaceutically acceptable salts thereof; about 2% toabout 16% by weight levodopa or an ester thereof; and about 0.6% toabout 1.4% by weight carbidopa or an ester thereof, and wherein themethod comprises administering less than 42 μg/day of hydrazine to thepatient.
 21. The method of claim 20, wherein the composition comprisesless than about 1 μg/ml of hydrazine, as determined by a GCMS method.22. The method of claim 20, wherein the composition comprises about 0.1μg/ml to about 2 μg/ml of hydrazine, as determined by a GCMS method. 23.The method of claim 20, wherein the composition after 30 days at −20°C., has less than about 0.2% by weight3,4-dihydroxyphenyl-2-methylpropionic acid, as determined by HPLC. 24.The method of claim 20, wherein the composition has less than about 5%or less than about 1% by weight of a degradant, relative to the amountof carbidopa or an ester thereof, as determined by HPLC.